VL1 Chromatin (I)

Question 1

What is the conundrum of DNA packaging?

Answer 1

Human DNA 2m long
Nucleus r = 10 μm

so we need 10,000 x compaction

Question 2

What is chromatin and how is it structured?

Answer 2

Chromatin organizes and compacts DNA, and resticts accessibility of the genome.
With long- range fiber-fiber intercation and short range internucleosomal interactions.

Subuinit of Chromatin is the Nucelosome:
* Approx. 147 bp of DNA wrapped around histone octamer, beads-on-a-string
* Each nucleosome consistsof 8 proteins, whch are incorporated as dimers (H2A/H2B and H3/H4).
2 H3/H4 dimers form the core
and H2A/H2B attach to the sides.

Question 3

Regulation of eukaryotics chromatin depends on?

Answer 3

• Histone modifications
• Histone variants
• Nucleosome remodeling
• DNA methylation

Question 4

What is the difference between heterochromatin and euchromatin?

Answer 4

Heterochromatin is densely packed and often transcriptionally inactive,

Euchromatin is less dense and more transcriptionally active.

Question 5

How does nucleosome positioning influence gene expression?

Answer 5

Nucleosome positioning regulates DNA accessibility to transcription machinery, with tightly positioned nucleosomes often repressing gene expression and well-spaced ones facilitating it.

Question 6

What are pioneer transcription factors?

Answer 6

Pioneer transcription factors can bind DNA sequences within nucleosomes, recruiting chromatin remodelers to open up chromatin for other transcription factors.

Question 7

What techniques are used to study nucleosome positioning?

Answer 7

MNase assay:
* MN (Micrococcal Nuclease) Enzyme that digest DNA
* MNase can only digest linker DNA (Located between nucleosomes, forming the “link” or spacer between the tightly wrapped nucleosomal DNA.Located in euchromatin and heterochromatin).
1. Isolate Nuclei: Obtain intact nuclei from cells.
2. Isolate Chromatin: Extract chromatin from nuclei.
3. Add MNase: Introduce enzyme to chromatin.
4. Inkubate: Incubate to allow digestion.
5. Stop Inkubation: At different times with stop solution.
6. DNA Extraction: Separate DNA from histones.
7. Gel Electrophoresis: Visualize DNA fragments. Nucleosome Ladder: Mononucleosomes (~147 bp), dinucleosomes (~300 bp), etc.
Note To identify which gene is on linker DNA and which on Histones use different gels. Mark DNA extraction with probe for youre gene.
8. Sequencing DNA

ChIP with Histone specific Antibodys
* ChIP is a technique used to analyze protein-DNA interactions.
1. Use cross-linking proteins to cross-link DNA to Histones
2. shearing the DNA into smaller pieces
3. add specific antibodies to modified histones
4. affinity purify histone-DNA-complexes
5. Remove cross-link
6. Examine by PCR, NGS or microarrays

DNase I hypersensitivity assay
* DNaseI can only access open chromatin (Refers to broader regions of the genome where the chromatin is in a relaxed, accessible state, typically associated with active regulatory elements.Located only in euchromatin)
solate Nuclei: Obtain intact nuclei from cells.
1. Isolate Chromatin: Extract chromatin from nuclei.
2. Add DNase I: Introduce enzyme to chromatin.
3. Inkubate: Incubate to allow digestion.
4. Stop Inkubation: At different times with stop solution.
5. DNA Extraction: Separate DNA from proteins.
6. Gel Electrophoresis: Visualize DNA fragments. –> Hypersensitive Sites: Smaller fragments indicating open chromatin regions.

Question 8

What is chromatin assembly?

Answer 8

Chromatin assembly is the process of organizing DNA into nucleosomes, involving histone chaperones and ATP-dependent remodelers, crucial during DNA replication, transcription, and repair.

Positioning can be very conserved/ these locations are maintained consistently across different cells or conditions